PROJECT SUMMARY Antimicrobial drug resistance is one of the biggest threats to public health globally. Developing new antibiotics alone cannot fully address the problem, as antimicrobial resistance will be developed eventually and inevitably to conventional antibiotics. Therefore, alternative non-antibiotic approaches are urgently needed to meet this global challenge. Antimicrobial photodynamic inactivation (PDI) is one of the alternatives. PDI utilizes light irradiation to excite photosensitizers in the presence of surrounding oxygen molecules to generate reactive oxygen species (ROS). The ROS can cause oxidative damage to various biological targets (e.g. proteins, lipids, and nucleic acids) non-specifically that leads to the ultimate cell death. PDI is a broad-spectrum antimicrobial approach that microbes are very unlikely to develop resistance against, and is excellent for the treatment of topical infections by nature. Still, PDI is currently hindered by the limited numbers of available photosensitizing molecules and their modest capabilities in ROS generation. Most photosensitizing molecules are hydrophobic and tend to aggregate in aqueous media, further reducing the ROS generation. There is, therefore, a critical need to develop photosensitizers dispersible in aqueous media while displaying high PDI efficacy and biocompatibility. Without such photosensitizers, the promise of PDI for clinical applications will likely remain unfulfilled. Previously, we have developed a type of novel silver-nanoparticle enhanced hybrid photosensitizers with high ROS generation and excellent PDI efficacy (up to ~6-log killing) on both gram-positive and gram- negative bacteria, including drug-resistant strains, while showing low photo-toxicity to primary cells under PDI conditions. Our innovation departs from the status quo by shifting the focus to hybrid photosensitizers displaying synergistic effect of silver nanoparticles and photosensitizing molecules. Our long-term goal is to translate these promising photosensitizers into effective treatment of topical infections. The objective of this Phase I proposal is to optimize and identify one or more formulations containing the hybrid photosensitizers with high PDI efficacy against biofilms, which are commonly associated with skin infections and resistant to antibiotics, and to validate their biocompatibility, in preparation for the in vivo animal testing in Phase II study. Our central hypothesis is that the identified formulations will exhibit high PDI efficacy against biofilms, while having low cytotoxicity on primary human cells in vitro and normal human skin explants ex vivo. This hypothesis will be tested by two specific aims: 1) Optimize semifluid formulations of the hybrid photosensitizers with high PDI efficacy against drug-resistant pathogens. 2) Validate the PDI efficacy of the optimized formulations against biofilms of the drug-resistant pathogens and establish the safety profile of the formulations in vitro and ex vivo. The rationale is that, results from this study will form the basis for subsequent efforts of animal tests and pre-clinical studies. The success of the study will substantively improve the effectiveness of PDI and promote its clinical uses, which will have an important positive impact in the conservation of essential antibiotics and alleviation of drug resistance.